The Iongterm goals of this project are to elucidate the multiple roles of Sindbis virus (SIN) RNA dependent RNA polymerase (RdRP) and how it differentially regulates the synthesis of the three major species of viral RNAs. Mosquito-transmitted RNA viruses are a leading cause of emereging infections worldwide. Alphaviruses including SIN and SIN-like viruses are transmitted by mosquitoes to animals and humans and cause periodic outbreaks of fever, arthritic syndrome, and encephalitis in animals and humans. Molecular and phylogentic studies indicate that highly pathogenic human alphaviruses such as Western Equine Encephalitis virus (WEE) have evolved from SIN by RNA recombination. Studies reported from the laboratories of the P.I and others have established that the viral RdRp plays a pivotal role in RNA recombination and generation of new viruses. The multiple functions of alphaviral polymerases (RdRp) and the RNA motifs that are recognized by them are poorly understood. Effective prevention of alphaviral diseases, and the increasing use of alpahvirus based gene and vaccine delivery vectors in biotechnology and medicine warrant detailed understanding of functions alphaviral RdRp. Alphavirus genomes are composed of a 12-kb (+) sense RNA that is capped and polyadenylated. Contrary to expectations, we have shown that a known 3' RNA motif is not required for initiation of RNA synthesis. On the basis of our published work, we hypothesize that one or more alternate RNA motifs other than the known 3' motif regulate viral (-) RNA synthesis. During this budget period, using SIN genomic and subgenomic replicons, we propose to investigate: a) the alternate promoter elements that are reponsible for (-) sense RNA synthesis; b) viral nonstructural proteins that interact with 3' promoter elements. Engineering SIN cDNA clones, RNA transfection studies into cultured cells, yeast two-hybrid and three-hybrid screening, and electrophoretic mobility shift assays will be used to address these specific aims.